Cellular and axonal transport phenotypes due to the C9ORF72 HRE in iPSC motor and sensory neurons

Summary Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) from patients with amyotrophic lateral sclerosis (ALS) and the C9ORF72 hexanucleotide repeat expansion (HRE) have multiple cellular phenotypes, but which of these accurately reflect the biology underlying the cell-specific vulnerability of ALS is uncertain. We therefore compared phenotypes due to the C9ORF72 HRE in MNs with sensory neurons (SNs), which are relatively spared in ALS. The iPSC models were able to partially reproduce the differential gene expression seen between adult SNs and MNs. We demonstrated that the typical hallmarks of C9ORF72-ALS, including RNA foci and dipeptide formation, as well as specific axonal transport defects, occurred equally in MNs and SNs, suggesting that these in vitro phenotypes are not sufficient to explain the cell-type selectivity of ALS in isolation.

On DIV 18, cells were dissociated with accutase, counted using trypan blue, and replated onto 0.07% polyethyleneimine (PEI; Merck)/Geltrex.A density of ~1,000,000 cells per well was used for 6-well plates and ~100,000 cells per well for 24-well plates.Basal media was supplemented with rock inhibitor, and γ-secretase inhibitor DAPT (10µM; Tocris Bioscience) was added to promote matura on.Media also contained growth factors BDNF and GDNF (both 10ng/ml; PreproTech).On DIV 19 cytosine arabinoside (AraC, 1µM; Sigma) was added.On DIV 23 AraC and DAPT were removed and mouse laminin (0.5µg/ml; ThermoFisher) was added for subsequent three mes weekly 50% media changes.Motor neuron pellets and coverslips were collected five weeks a er final pla ng (DIV 53).A schema c of the differen a on can be found in Figure 1A.
On DIV 11 cells were dissociated with TrypLE (ThermoFisher), counted and replated onto PEI/Geltrex coated plates as described for MNs.SU and DAPT were removed and media was supplemented with growth factors NGF (25ng/ml; Peprotech), BDNF (25ng/ml), GDNF (25ng/ml) and Neurotrophin-3 (25ng/ml; Peprotech).As for MNs, rock inhibitor was added on the DIV of spli ng and AraC was present between DIV 11 and 13.On DIV 13 AraC was removed and laminin was added to the media.From DIV 13 to media changes (50%) were performed three mes a week.From around DIV 30 the concentra on of all four growth factors was reduced to 10ng/ml.Sensory neuron pellets and coverslips were collected five weeks a er final pla ng (DIV 45).A schema c of the differen a on can be found in Figure 1A.

Protein extraction and Quantification
A er a wash with PBS, cells were collected in a small volume of phosphate buffer saline (PBS) and pelleted at 3000rpm for 5 minutes at 4°C.Cells were lysed in RIPA buffer (Sigma) and protease inhibitors (Roche) following mechanically homogenisa on.A er 30 minutes lysates were centrifuged at 10,000rpm for 5 minutes at 4°C and the pellets were discarded.Total protein concentra on of the supernatant was determined using the bicinchoninic acid assay (Sigma) according to the manufacturer's instruc ons.

Western blotting
For gel electrophoresis, protein in RIPA buffer was mixed with NuPAGE LDS sample buffer and NuPAGE sample reducing agent and heated to 95°C for 5 minutes, except where indicated.10µg per well was loaded on NuPAGE 4-12% Bis-Tris gels (ThermoFisher) and then run at 100V for 120 minutes.Resolved protein was transferred to nitrocellulose membranes using the iBlot 2 Gel Transfer Device (ThermoFisher).Blots were blocked for 1 hour at room temperature in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% skimmed milk and incubated overnight at 4°C with primary an bodies (Table 2) in TBS + 0.1% Tween-20 + 1% skimmed milk.Blots were washed three mes and incubated with secondary an body (Supplementary Table 3) in TBS + 0.1% Tween-20 + 1% skimmed milk for 1 hour at room temperature.Blots were washed a further three mes before visualisa on on a Bio-Rad ChemiDoc.Integrated op cal density of the protein bands was measured using Fiji and normalised to β-ac n for the same sample.

Immunocytochemistry
Cells grown on glass coverslips were fixed in 4% methanol-free paraformaldehyde for 30 minutes and washed three mes with PBS.Coverslips were blocked and permeabilised (0.1% Triton-X; Dow, and 5% donkey serum; BioRad, in PBS) for 1 hour and then incubated overnight with primary an body in an body buffer (0.1% Triton-X and 1% donkey serum in PBS).Samples were rinsed in wash buffer (0.1% Triton-X in PBS) for ten minutes and then incubated with secondary an bodies in an body buffer for one hour.Coverslips were washed again and then incubated with 4',6-Diamidino-2-Phenylindole (DAPI, 1:10,000 dilu on in PBS) for ten minutes, followed by a PBS-only wash.Coverslips were mounted in Prolong Diamond An fade mountant (ThermoFisher).

DPR extraction and ELISA
Frozen cell pellets were lysed using RIPA buffer containing an EDTA-free protease inhibitor cocktail (2x, Sigma) and 10% sodium dodecyl sulphate (SDS, ThermoFisher) and sonicated at 30% AMP for 3 x 10 seconds with 5 s-intervals.The probe was cleaned with ethanol between samples.Samples were centrifuged at 17,000xg for 20 minutes at 16°C and the pellets were discarded.Total protein concentra on of the supernatant was determined using a BCA assay and all samples were adjusted to 0.5mg/ml.Plates were coated with unlabelled an -poly (GP) or an -poly (GA) an bodies.A er blocking, samples were loaded at 45 µg of protein per well for poly (GP) and 27 µg for poly (GA).Bio nylated an -poly (GP) and an -poly (GA) an bodies were used as detectors, followed by sulfotagged streptavidin (Meso Scale Discovery, R32AD).Plates were read with the MSD reading buffer (Meso Scale Discovery, R92TC) using the MSD Sector Imager 2400.Signals correspond to intensity of emi ed light upon electrochemical s mula on of the assay plate.Prior to analysis, the average reading from a calibrator containing no pep de was subtracted from each reading.

Fluorescence in situ hybridisation
MNs and SNs were fixed in 4% paraformaldehyde for 30 minutes and washed three mes with PBS.All work was carried out in an RNase free environment, using RNase ZAP (ThermoFisher).Cells were permeabilised with 0.1% Triton-X and then washed three mes in PBS.Tissue was dehydrated with serial addi on of 70%, 90% and 100% ethanol and then allowed to air dry before rehydra on in PBS followed by 2x Standard Sodium Citrate (SSC) buffer (Sigma).Coverslips were incubated at 80°C for 45 minutes with pre-hybridisa on solu on (50% Formamide; ThermoFisher and 2x SSC in dis lled water) followed by two hours in hybridisa on solu on (2x SSC, 0.016% BSA; ThermoFisher, 0.8mg/ml salmon sperm DNA; ThermoFisher, 0.8 mg/ml yeast tRNA; ThermoFisher, 8% Dextran sulphate; Amresco, 50% Formamide, 1.6 mM Vanadyl Ribonucleo de; BioLabs, 5mM EDTA and 0.2ng/µl Cy-3conjugated 2'O-methyl sense oligonucleo de RNA probe; Integrated DNA Technologies) and coverslips were incubated at 80°C for 2 hours.Coverslips were washed three mes for 30 minutes each in washing buffer (50% formamide, 0.5x SSC in dis lled water).The hybridisa on solu on was then made up again but using the AlexaFluor488-conjugated an sense oligonucleo de probe (0.2ng/µl) and coverslips were incubated at 80°C for a further 2 hours, followed by three washes in washing buffer as before.Coverslips were washed for 10 minutes three mes in 0.5x SSC and finally washed once with PBS.Samples were blocked for 30 minutes (10% donkey serum, 0.1% Triton-X in PBS) and stained with DAPI and Tuj1 according to the immunofluorescence protocol described above.

Manufacture of microfluidic devices
PDMS prepolymer and catalyst (10:1) were mixed thoroughly and poured into a master mould, and subsequently cured in an oven for 2h at 40 °C.Cured PDMS was removed from the master mould and 8 mm reservoirs were punched out.Irreversible bonding was achieved using a plasma cleaner.

Table 4 :
Supplementary Table1: iPSC lines used in this study.Clones from the same pa ent are grouped.Abbrevia ons: PMID = PubMed iden fica on number; ALS = amyotrophic lateral sclerosis Growth factors used during differen a on.